1. Field of the Invention
The present invention relates to the construction of altered DNA molecules utilizing polymerase chain reaction. The alteration may involve insertion, deletion, repetition (both direct and inverted), or substitution of DNA sequences with a high degree of precision and may be accomplished in certain instances for alterations as small as a single base pair. The invention relates as well to the construction of chimeric DNA molecules.
2. Description of the Related Art
The polymerase chain reaction (PCR) technique was conceived and developed by the Cetus Corporation to provide for specific amplification of discrete fragments of DNA in order to allow simplified detection and purification of nucleic acid fragments initially present in a particular sample in only picogram quantities (Saiki, et al., Science 230:1350-1354, 1985). The procedure mimics the natural DNA replication process in that the number of DNA molecules generated doubles after each cycle. The basic method is based on the repetition of three steps, all conducted in a successive fashion under controlled temperature conditions: (1) denaturing the double-stranded template DNA; (2) annealing the single-stranded primers to the complementary single-stranded regions on the template DNA; and, (3) synthesizing additional DNA along the templates by extension of the primer DNAs with DNA polymerase. After 4 to 25 cycles of these steps, as much as a 10.sup.5 -fold increase in the original DNA is observed (Oste, BioTechniques 6:162-167, 1988).
Initially, the PCR technique was used primarily in cloning and sequencing applications. More recently, PCR technology has been used for mutagenesis of specific DNA sequences and for other directed manipulations of DNA.
For instance, PCR technology has been used to engineer hybrid (chimeric) genes without the need to use restriction enzymes in order to segment the gene prior to hybrid formation. In this approach, fragments of the different genes that are to form the hybrid are generated in separate polymerase chain reactions. The primers used in these separate reactions are designed so that the ends of the different products of the separate reactions contain complementary sequences. When these separately produced PCR products are mixed, denatured and reannealed, the strands having matching sequences at their 3'-ends overlap and act as primers for each other. Extension of this overlap by DNA polymerase produces a molecule in which the original sequences are spliced together to form the hybrid gene. Thus, this method requires four primers to construct a deleted, hybrid DNA molecule. Likewise, the method requires six primers and three rounds of PCR in order to construct a chimeric molecule. Furthermore, it does not allow a straightforward means to generate inverted or directly repeated DNA sequences (Horton, et al., Gene 77:61-68, 1989).
Since the primer regions used for PCR need not match the template gene sequence exactly, it has been possible to incorporate restriction sites within the primers (Scharf, et al., Science 233:1076-1078, 1986). A recent application of this approach involved the cloning and expression of immunoglobulin V genes (Orlandi, et al., Proc.Natl.Acad.Sci. 86:3833-3837, 1989). In this application of PCR technology, use was made of the conserved regions at each end of the nucleotide sequences encoding the V domains of mouse immunoglobulin heavy chain (V.sub.H) and light chain (V.sub.K). Primers were designed for PCR amplification which incorporated restriction sites and which, thereby, mutated the PCR-generated fragments to allow for ease in subcloning the amplified regions into appropriate expression vectors.
In other applications, it was possible to vary the standard PCR approach (which requires oligonucleotide primers complementary to both ends of the segment to be amplified) to allow amplification of DNA flanked on only one side by a region of known DNA sequence (Silver and Keerikatte, J.Virol. 63:1924-1928, 1989). This technique requires the presence of a known restriction site within the known DNA sequence and a similar site within the unknown flanking DNA sequence which is to be amplified. After restriction and recircularization, the recircularized fragment is restricted at an unique site between the two primers and the resulting linearized fragment is used as a template for PCR amplification.
Another approach which allows the amplification of unknown sequences of DNA was developed by Triglia, et al. (Nucl.Acids Res. 16:8186, 1988). The approach requires the inversion of the sequence of interest by circularization and re-opening at a site distinct from the one of interest, and is called "inverted PCR." A fragment is first created in which two unknown sequences flank on either side a region of known DNA sequence. The fragment is then circularized and cleaved with an unique restriction endonuclease which only cuts within the known DNA sequence creating a new fragment containing all of the DNA of the original fragment but which is then inverted with regions of known sequence flanking the region of unknown sequence. This fragment is then utilized as a PCR substrate to amplify the unknown sequence.
Mutant and chimeric genes have also been produced using PCR in a specific approach which involves using a supercoiled plasmid DNA as a template for PCR and a primer bearing some sort of mutated sequence which is incorporated into the amplified product. Using this procedure, a single amino acid replacement, a sixteen amino acid deletion and a twelve amino acid substitution were introduced into the terminal signal sequence of rat hepatic cytochrome P450b (Vallette, et al., Nucl.Acids Res. 17:723-733, 1989). Using the method of this reference DNA sequences may be inserted only at the 5'-end of the DNA molecule which one wishes to alter. In addition, since the inserted sequence is part of the primer and has to be synthesized, the technique is limited to shorter DNA sequences amenable to economical DNA synthesis (synthetic DNAs over 100 bp are very expensive). It is not clear how hybrid or substituted DNA molecules can be constructed by this technique.
In a procedure similar to that of Vallette, et al. (1989), PCR was used to create deletions within existing expression plasmids (Mole, Nucl.Acids Res. 17:3319, 1989). This application of PCR technology demonstrated the advantage that the modified region need not be excised from the plasmid. However, PCR was performed around the entire plasmid (containing the fragment to be deleted) from primers whose 5'-ends defined the region to be deleted. Self-ligation of the PCR product recircularized the plasmid. Recircularization required by this method, however, is typically low efficiency and the yield of recircularized DNA is hard to control.
One of the major limitations of PCR technology as currently practiced in the construction of DNA molecules is the error-prone nature of Tag polymerase. Use of Tag polymerase results on average in one mutation per 400 base pairs polymerized per 30 PCR cycles. Thus, the higher the number of cycles used to produce the desired PCR product, the higher the probability of generating unwanted mutations. Such mutations are especially critical where functional DNA products such as protein coding regions or promoter domains are very sensitive to change.
The techniques of the prior art (supra) typically require the introduction of mutated primers or require extensive DNA duplication around the entire length of a plasmid vector in order to introduce deletions, insertions or substitutions into a specific site within a given DNA sequence. Furthermore, certain of the prior art approaches require multiple primers and multiple PCR-treatments in order to achieve the desired alteration. Some of the methods taught by the prior art are limited to alterations at the termini of a DNA molecule and others require inefficient recircularization of the vector carrying the DNA sequence to be altered. Still others are limited by the size of economical synthetic primers. None of the prior art references appear to be readily amenable to generation of repeated sequences within a DNA molecule. A universally applicable method which utilizes the advantages of PCR amplification but which is not limited in the manners outlined above is needed to make PCR-generated DNA alteration a generally useful tool.